Growth differentiation factor 15 fusion proteins

ABSTRACT

GDF15 molecules are provided herein. In some embodiments, the GDF15 molecule is a GDF15-Fc fusion, in which a GDF15 region is fused to an Fc region. In some embodiments, the GDF15 region is fused to the Fc region via a linker. Also, provided herein are methods for making and using GDF15 molecules.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/620,029, filed on Dec. 6, 2019, which is a U.S. national stage filingunder 35 U.S.C. § 371 of PCT Application No. PCT/US2019/026369, filed onApr. 8, 2019, which claims the benefit of U.S. Provisional ApplicationNo. 62/655,108, filed on Apr. 9, 2018, which are all hereby incorporatedby reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledA-2239-US-CNT_SeqList.txt, created Jan. 8, 2021, which is 109 kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The instant disclosure relates to GDF15 molecules, such as GDF15 fusionproteins, compositions thereof, and methods for making and using suchproteins.

BACKGROUND

Growth differentiation factor 15 (GDF15), also referred to as macrophageinhibitory cytokine 1 (MIC1) (Bootcov M R, 1997, Proc Natl Acad Sci94:11514-9), placental bone morphogenetic factor (PLAB) (Hromas R 1997,Biochim Biophys Acta. 1354:40-4), placental transforming growth factorbeta (PTGFB) (Lawton L N 1997, Gene. 203:17-26), prostate derived factor(PDF) (Paralkar V M 1998, J Biol Chem. 273:13760-7), and nonsteroidalanti-inflammatory drug-activated gene (NAG-1) (Baek S J 2001, J BiolChem. 276: 33384-92), is a secreted protein that circulates in plasma asan ˜25 kDa homodimer. GDF15 binds to GDNF family receptor α-like (GFRAL)with high affinity. GDF15-induced cell signaling is believed to requirethe interaction of GFRAL with the coreceptor RET.

GDF15 has been linked to multiple biological activities. Elevated GDF15has been shown to be correlated with weight loss and administration ofGDF15 has been shown to reduce food intake and body weight. Accordingly,there is a need for efficacious GDF15 molecules that can be administeredas a therapeutic. The present disclosure provides GDF15 molecules thatmeets this need and provide related advantages.

SUMMARY

Provided herein are GDF15 molecules, methods of making the molecules andmethods of using the molecules. In some embodiments, the GDF15 moleculeis a GDF15-Fc fusion protein. The fusion protein can comprise a GDF15region joined to an Fc region. In some embodiments, the GDF15 region isjoined to the Fc via a linker.

In some embodiments, the GDF15 region comprises the amino acid sequenceof SEQ ID NO: 6 and at least one mutation, such as a mutation of theasparagine at position 3 (N3), as a mutation of the aspartate atposition 5 (D5), or mutations of the asparagine at position 3 and theaspartate at position 5. In some embodiments, the GDF15 region comprisesa mutation of the aspartate at position 5 to glutamate (D5E). In someembodiments, the GDF15 region comprises the amino acid sequence of SEQID NO: 16. In some embodiments, the GDF15 region comprises a mutation ofthe asparagine at position 3 to glutamine (N3Q), for example, having anamino acid sequence SEQ ID NO: 14. In yet other embodiments, the GDF15region comprises both N3Q and D5E mutations. In some embodiments, theGDF15 region comprises the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the fusion protein has a linker that is a G4S (SEQID NO: 19) or G4Q (SEQ ID NO: 24) linker, such as a (G4S)n or (G4Q)nlinker, wherein n is greater than 0. In some embodiments, the fusionprotein has a linker that is a G4A (SEQ ID NO: 58) linker, such as a(G4A)n linker, wherein n is greater than 0. In some embodiments, n is 1or 2. In some embodiments, n is greater than 2, such as 3, 4, 5, 6, 7,or 8. In some embodiments, the linker comprises the amino acid sequenceof SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, or 58.

In some embodiments, the fusion protein has an Fc region comprises acharged pair mutation. In some embodiments, the Fc region has atruncated hinge region. In some embodiments, the Fc region is selectedfrom Table 3.

Also provided herein are dimers and tetramers comprising the fusionproteins disclosed herein. In one embodiment, the dimer comprises aGDF15-Fc fusion comprising the amino acid sequence of any one of SEQ IDNOs: 39-57. In some embodiments, a GDF15-Fc fusion comprising the aminoacid sequence of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56 or 57 dimerizes with an Fc domain comprisingthe amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, or 37, such asshown in Table 6. In some embodiments, the dimers form tetramers.Methods of producing and using the GDF15 molecules disclosed herein arealso provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect on the body weight of cynomologusmonkeys dosed with vehicle, 3 mg/kg of the positive control FGF21-Fc,1.5 mg/kg of scFc-GDF15, or 1.5 mg/kg of the dimerFcΔ10(−)-(G4S)4-GDF15:FcΔ10(+,K) weekly for six weeks, followed by afive-week washout.

FIG. 2 is a plot showing the effect on the triglyceride levels ofcynomologus monkeys dosed with vehicle, 3 mg/kg of the positive controlFGF21-Fc, 1.5 mg/kg of scFc-GDF15, or 1.5 mg/kg of the dimerFcΔ10(−)-(G4S)4-GDF15:FcΔ10(+,K) weekly for six weeks, followed by afive-week washout.

FIG. 3 shows the profile of FcΔ10(−)-(G4S)4-GDF15 after cation exchange.

FIG. 4 is a peptide map of FcΔ10(+)-(G4)-GDF15.

FIG. 5 is a graph showing the effect on food intake in mice as afunction of dose of the dimers FcΔ10(−)-GDF15(Δ3):FcΔ10(+,K) (SEQ IDNOs: 41 and 32); FcΔ10(−)-GDF15(N3D):FcΔ10(+,K) (SEQ ID NOs: 42 and 32);FcΔ10(−,CC)-GDF15(Δ3):FcΔ10(+,K,CC) (SEQ ID NOs: 43 and 34);FcΔ10(−,CC)-GDF15(N3D):FcΔ10(+,K,CC) (SEQ ID NOs: 44 and 34)) andFcΔ10(−)-(G45)4-GDF15:FcΔ10(+,K) (SEQ ID NOs:39 and 32).

FIG. 6 is a graph of the serum concentration of FcΔ10(−)-GDF15(Δ3) (SEQID NO: 41); FcΔ10(−)-GDF15(N3D) (SEQ ID NO: 42); FcΔ10(−,CC)-GDF15(Δ3)(SEQ ID NO: 43); and FcΔ10(−,CC)-GDF15(N3D) (SEQ ID NO: 44)) as afunction of time in mice.

FIG. 7 is a graph showing the effect on the body weight of cynomologusmonkeys dosed with vehicle, 3 mg/kg of the positive control FGF21-Fc,0.5 mg/kg or 3.0 mg/kg of FcΔ16(−,CC)-GDF15(Δ3/D5E):FcΔ16(+,K,CC) (SEQID NOs: 45 and 35), 0.5 mg/kg or 3.0 mg/kg ofFcΔ16(−,CC)-GDF15(N3Q/D5E):FcΔ16(+,K,CC)) (SEQ ID NOs: 46 and 35) or 0.5mg/kg or 3.0 mg/kg of FcΔ16(−)-GDF15(N3Q/D5E):FcΔ16(+,K) (SEQ ID NOs: 47and 36) weekly for four weeks, followed by a four-week washout.

FIG. 8 is a graph showing the effect on the body weight of cynomologusmonkeys dosed with vehicle, 1.5 mg/kg ofFcΔ10(−)-(G45)4-GDF15:FcΔ10(+,K) (SEQ ID NOs: 39 and 32), 1.5 mg/kg ofFcΔ16(−)-(G4Q)4-GDF15(N3Q):FcΔ16(+,K) (SEQ ID NOs: 49 and 36); 1.5 mg/kgof FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E):FcΔ16(+,K) (SEQ ID NOs: 50 and 36),1.5 mg/kg of FcΔ16(−)-G4S-GDF15(N3Q/D5E):FcΔ16(+,K) (SEQ ID NOs: 54 and36), or 1.5 mg/kg ofFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):FcΔ10(+,K,L234A/L235A) (SEQID NOs: 57 and 37) weekly for two weeks.

FIG. 9 is a graph of food intake as a function of dose in ob/ob miceadministered FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E):FcΔ16(+,K) (SEQ ID NOs: 50and 36).

FIG. 10 is a graph of food intake as a function of dose in ob/ob miceadministeredFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):FcΔ10(+,K,L234A/L235A) (SEQID NOs: 57 and 37).

DETAILED DESCRIPTION

Provided herein are GDF15 molecules, methods of making the molecules andmethods of using the molecules. In some embodiments, the GDF15 moleculeis a GDF15-Fc fusion protein. The fusion protein can comprise a GDF15region joined to an Fc region. In some embodiments, the GDF15 region isjoined to the Fc via a linker.

In some embodiments, the GDF15 region comprises wild type GDF15. Boththe human and murine GDF15 have a signal peptide and prodomain. Thenucleotide sequence for full-length human GDF15 is:

(SEQ ID NO: 1) atgcccgggc aagaactcag gacggtgaat ggctctcagatgctcctggt gttgctggtg ctctcgtggc tgccgcatgggggcgccctg tctctggccg aggcgagccg cgcaagtttcccgggaccct cagagttgca ctccgaagac tccagattccgagagttgcg gaaacgctac gaggacctgc taaccaggctgcgggccaac cagagctggg aagattcgaa caccgacctcgtcccggccc ctgcagtccg gatactcacg ccagaagtgcggctgggatc cggcggccac ctgcacctgc gtatctctcgggccgccctt cccgaggggc tccccgaggc ctcccgccttcaccgggctc tgttccggct gtccccgacg gcgtcaaggtcgtgggacgt gacacgaccg ctgcggcgtc agctcagccttgcaagaccc caggcgcccg cgctgcacct gcgactgtcgccgccgccgt cgcagtcgga ccaactgctg gcagaatcttcgtccgcacg gccccagctg gagttgcact tgcggccgcaagccgccagg gggcgccgca gagcgcgtgc gcgcaacggggaccactgtc cgctcgggcc cgggcgttgc tgccgtctgcacacggtccg cgcgtcgctg gaagacctgg gctgggccgattgggtgctg tcgccacggg aggtgcaagt gaccatgtgcatcggcgcgt gcccgagcca gttccgggcg gcaaacatgcacgcgcagat caagacgagc ctgcaccgcc tgaagcccgacacggtgcca gcgccctgct gcgtgcccgc cagctacaatcccatggtgc tcattcaaaa gaccgacacc ggggtgtcgctccagaccta tgatgacttg ttagccaaag actgccactg catatga

The amino acid sequence for full-length human GDF15 (308 amino acids)is:

(SEQ ID NO: 2) MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL LAKDCHCI

The nucleotide sequence for human GDF15 without its signal sequence is:

(SEQ ID NO: 3) ctgtctctgg ccgaggcgag ccgcgcaagt ttcccgggaccctcagagtt gcactccgaa gactccagat tccgagagttgcggaaacgc tacgaggacc tgctaaccag gctgcgggccaaccagagct gggaagattc gaacaccgac ctcgtcccggcccctgcagt ccggatactc acgccagaag tgcggctgggatccggcggc cacctgcacc tgcgtatctc tcgggccgcccttcccgagg ggctccccga ggcctcccgc cttcaccgggctctgttccg gctgtccccg acggcgtcaa ggtcgtgggacgtgacacga ccgctgcggc gtcagctcag ccttgcaagaccccaggcgc ccgcgctgca cctgcgactg tcgccgccgccgtcgcagtc ggaccaactg ctggcagaat cttcgtccgcacggccccag ctggagttgc acttgcggcc gcaagccgccagggggcgcc gcagagcgcg tgcgcgcaac ggggaccactgtccgctcgg gcccgggcgt tgctgccgtc tgcacacggtccgcgcgtcg ctggaagacc tgggctgggc cgattgggtgctgtcgccac gggaggtgca agtgaccatg tgcatcggcgcgtgcccgag ccagttccgg gcggcaaaca tgcacgcgcagatcaagacg agcctgcacc gcctgaagcc cgacacggtgccagcgccct gctgcgtgcc cgccagctac aatcccatggtgctcattca aaagaccgac accggggtgt cgctccagacctatgatgac ttgttagcca aagactgcca ctgcatatga

The amino acid sequence for human GDF15 without its 29 amino acid signalsequence (279 amino acids) is:

(SEQ ID NO: 4) LSLAEASRASFPGPSELHSEDSRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

The nucleotide sequence for human GDF15 without its signal peptide orprodomain is:

(SEQ ID NO: 5) gcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcatatga

The amino acid sequence for human GDF15 without its signal peptide orpro-domain (the active domain of GDF15 of 112 amino acids) is:

(SEQ ID NO: 6) ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

The nucleotide sequence for full-length murine GDF15 is:

(SEQ ID NO: 7) atggccccgc ccgcgctcca ggcccagcct ccaggcggctctcaactgag gttcctgctg ttcctgctgc tgttgctgctgctgctgtca tggccatcgc agggggacgc cctggcaatgcctgaacagc gaccctccgg ccctgagtcc caactcaacgccgacgagct acggggtcgc ttccaggacc tgctgagccggctgcatgcc aaccagagcc gagaggactc gaactcagaaccaagtcctg acccagctgt ccggatactc agtccagaggtgagattggg gtcccacggc cagctgctac tccgcgtcaaccgggcgtcg ctgagtcagg gtctccccga agcctaccgcgtgcaccgag cgctgctcct gctgacgccg acggcccgcccctgggacat cactaggccc ctgaagcgtg cgctcagcctccggggaccc cgtgctcccg cattacgcct gcgcctgacgccgcctccgg acctggctat gctgccctct ggcggcacgcagctggaact gcgcttacgg gtagccgccg gcagggggcgccgaagcgcg catgcgcacc caagagactc gtgcccactgggtccggggc gctgctgtca cttggagact gtgcaggcaactcttgaaga cttgggctgg agcgactggg tgctgtccccgcgccagctg cagctgagca tgtgcgtggg cgagtgtccccacctgtatc gctccgcgaa cacgcatgcg cagatcaaagcacgcctgca tggcctgcag cctgacaagg tgcctgccccgtgctgtgtc ccctccagct acaccccggt ggttcttatgcacaggacag acagtggtgt gtcactgcag acttatgatgacctggtggc ccggggctgc cactgcgctt ga

The amino acid sequence for full-length murine GDF15 (303 amino acids)is:

(SEQ ID NO: 8) MAPPALQAQPPGGSQLRFLLFLLLLLLLLSWPSQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVSLQTYDDLVARGC HCA

The nucleotide sequence for murine GDF15 without its signal sequence is:

(SEQ ID NO: 9) tcgcagggggacgccctggcaatgcctgaacagcgaccctccggccctgagtcccaactcaacgccgacgagctacggggtcgcttccaggacctgctgagccggctgcatgccaaccagagccgagaggactcgaactcagaaccaagtcctgacccagctgtccggatactcagtccagaggtgagattggggtcccacggccagctgctactccgcgtcaaccgggcgtcgctgagtcagggtctccccgaagcctaccgcgtgcaccgagcgctgctcctgctgacgccgacggcccgcccctgggacatcactaggcccctgaagcgtgcgctcagcctccggggaccccgtgctcccgcattacgcctgcgcctgacgccgcctccggacctggctatgctgccctctggcggcacgcagctggaactgcgcttacgggtagccgccggcagggggcgccgaagcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttgaagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacagacagtggtgtgtcactgcagacttatgatgacctggtggcccgggg ctgccactgcgcttga

The amino acid sequence for murine GDF15 without its 32 amino acidsignal sequence (271 amino acids) is:

(SEQ ID NO: 10) SQGDALAMPEQRPSGPESQLNADELRGRFQDLLSRLHANQSREDSNSEPSPDPAVRILSPEVRLGSHGQLLLRVNRASLSQGLPEAYRVHRALLLLTPTARPWDITRPLKRALSLRGPRAPALRLRLTPPPDLAMLPSGGTQLELRLRVAAGRGRRSAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHR TDSGVSLQTYDDLVARGCHCA

The nucleotide sequence for murine GDF15 without its signal sequence orpro-domain is:

(SEQ ID NO: 11) agcgcgcatgcgcacccaagagactcgtgcccactgggtccggggcgctgctgtcacttggagactgtgcaggcaactcttgaagacttgggctggagcgactgggtgctgtccccgcgccagctgcagctgagcatgtgcgtgggcgagtgtccccacctgtatcgctccgcgaacacgcatgcgcagatcaaagcacgcctgcatggcctgcagcctgacaaggtgcctgccccgtgctgtgtcccctccagctacaccccggtggttcttatgcacaggacagacagtggtgtgtcactgcagacttatgatgacctggtggcccggggctgccactgcgcttga

The amino acid sequence for murine GDF15 without its signal peptide orprodomain (active domain of 115 amino acids) is:

(SEQ ID NO: 12) SAHAHPRDSCPLGPGRCCHLETVQATLEDLGWSDWVLSPRQLQLSMCVGECPHLYRSANTHAQIKARLHGLQPDKVPAPCCVPSSYTPVVLMHRTDSGVS LQTYDDLVARGCHCA

In some embodiments, the GDF15 molecule comprises a GDF15 regioncomprising an active domain of GDF15, e.g., GDF15 without its signalpeptide or pro-domain. In some embodiments, the GDF15 region comprisesthe amino acid sequence of SEQ ID NO: 6 or 12. In some embodiments, theGDF15 region comprises a GDF15 sequence with one or more mutations, suchas at least one mutation in the active domain of GDF15. In particularembodiments, the mutation or mutations do not reduce or eliminate theactivity of GDF15. In some embodiments, the GDF15 region comprises amutation in the active domain of human GDF15. In one embodiment, themutation is a deletion of the first three amino acids of the activedomain, such as “GDF15(Δ3)” which is an active domain of human GDF15 inwhich the first three amino acids removed (i.e., SEQ ID NO: 13).

In some embodiments, the GDF15 region comprises a mutation of theasparagine at position 3 (N3) of the active domain of human GDF15 (SEQID NO: 6). An N3 mutation can refer to the mutation of the asparagineresidue at position 3 of SEQ ID NO: 6 or the mutation of an asparagineresidue corresponding to the asparagine at position 3 of SEQ ID NO: 6 ina GDF15 amino acid sequence. In some embodiments, the asparagine atposition 3 is mutated to glutamine (N3Q) or aspartate (N3D).Accordingly, in some embodiments, the GDF15 molecule comprises a GDF15region of GDF15(N3Q), which has the amino acid sequence of SEQ ID NO:14. In other embodiments, the GDF15 molecule comprises a GDF15 region ofGDF15(N3D), which has the amino acid sequence of SEQ ID NO: 15. In someembodiments, the GDF15 region comprises a mutation of the aspartate atposition 5 (D5) of the active domain of human GDF15 (SEQ ID NO: 6). A D5mutation can refer to the mutation of the aspartate residue at position5 of SEQ ID NO: 6 or the mutation of an aspartate residue correspondingto the aspartate at position 5 of SEQ ID NO: 6 in a GDF15 amino acidsequence. In one embodiment, the aspartate at position 5 is mutated toglutamate (D5E). Accordingly, in some embodiments, the GDF15 moleculecomprises a GDF15 region of GDF15(D5E), which has the amino acidsequence of SEQ ID NO: 16.

In yet other embodiments, the GDF15 region comprises a combination ofmutations, such as a combination of Δ3 and D5 mutations, e.g.,GDF15(Δ3/D5E) (SEQ ID NO: 17) or a combination of N3 and D5 mutations,e.g., GDF15(N3D/D5E) or GDF15(N3Q/D5E). In, the GDF15 region comprisesthe amino acid sequence of SEQ ID NO: 18.

Table 1 provides examples of GDF15 regions that can be used in the GDF15molecules.

TABLE 1 GDF15 Regions SEQ ID NO: Designation Sequence 6 GDF15ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLS PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI 13 GDF15(Δ3)GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPRE VQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDC HCIFirst third amino acids at N-terminus of GDF15 sequence(SEQ ID NO: 6) is deleted in this GDF15 region. 14 GDF15(N3Q) AR QGDHCPLGPGRCCRLHTVRASLEDLGWADWVLS PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCIUnderlined and bolded residue is N3Q mutation. 15 GDF15(N3D) AR DGDHCPLGPGRCCRLHTVRASLEDLGWADWVLS PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCIUnderlined and bolded residue is N3D mutation. 16 GDF15(D5E) ARNG EHCPLGPGRCCRLHTVRASLEDLGWADWVLS PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCIUnderlined and bolded residue is D5E mutation. 17 GDF15(Δ3/D5E) G EHCPLGPGRCCRLHTVRASLEDLGWADWVLSPRE VQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDC HCIFirst third amino acids at N-terminus of GDF15 sequence(SEQ ID NO: 6) is deleted in this GDF15 region;underlined and bolded residue is D5E mutation (position inreference to wild-type GDF15 sequence of SEQ ID NO: 6). 18GDF15(N3Q/D5E) AR Q G E HCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCIUnderlined and bolded residues are N3Q and D5E mutations.

In some embodiments, the GDF15 molecule is fused to an Fc directly. Inother embodiments, the Fc is fused to the GDF15 molecule via a linker.In some embodiments, the linker comprises a G4S (SEQ ID NO: 19) linker.In other embodiments, the linker comprises a G4Q (SEQ ID NO: 24) linker.In other embodiments, the linker comprises a G4A (SEQ ID NO: 58) linker.The linker can be a (G4S)n or (G4Q)n linker, wherein n is greater than0. The linker can be a (G4A)n linker, wherein n is greater than 0. Insome embodiments, n is 1 or 2. In some embodiments, n is greater than orequal to 2, such as 3, 4, 5, 6, 7, or 8. In some embodiments, the linkercomprises the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24,25, or 58 as shown in Table 2.

TABLE 2 Linkers SEQ ID NO: Designation Sequence 19 G4S GGGGS 20 (G4S)2GGGGSGGGGS 21 (G4S)4 GGGGSGGGGSGGGGSGGGGS 22 (G4S)8GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGS 23 G4 GGGG 24 G4Q GGGGQ 25(G4Q)4 GGGGQGGGGQGGGGQGGGGQ 58 G4A GGGGA

In some embodiments, the GDF15 molecule comprises an Fc region. The Fcregion can comprise or be derived from the Fc domain of a heavy chain ofan antibody. In some embodiments, the Fc region may comprise an Fcdomain with a mutation, such as a charged pair mutation, a mutation in aglycosylation site or the inclusion of an unnatural amino acid. The Fcregion can be derived from a human IgG constant domain of IgG1, IgG2,IgG3 or IgG4. In some embodiments, the Fc region comprises the constantdomain of an IgA, IgD, IgE, and IgM heavy chain.

In some embodiments, the Fc region comprises an Fc domain with a chargedpair mutation. By introducing a mutation resulting in a charged Fcregion, the GDF15 molecule can dimerize with a corresponding Fc moleculehaving the opposite charge. For example, an aspartate-to-lysine mutation(E356K, wherein 356 is the position using EU numbering, and correspondsto the positions as noted in Tables 3-5) and a glutamate-to-lysinemutation (D399K wherein 399 is the position using EU numbering, andcorresponds to positions as noted in Tables 3-5) can be introduced intothe Fc region that is joined to a GDF15 region, optionally via a linker,resulting in a positively charged Fc region for the GDF15 molecule.Lysine-to-aspartate mutations (K392D, K409D; wherein 392 and 409 are thepositions using EU numbering and corresponds to the positions as notedin Tables 3-5) can be introduced into an Fc domain of a separatemolecule, resulting in a negatively charged Fc molecule. The aspartateresidues in the negatively charged Fc molecule can associate with thelysine residues of the positively charged Fc region of the GDF15molecule through electrostatic force, facilitating formation of Fcheterodimers between the Fc region of the GDF15 molecule and the Fcmolecule, while reducing or preventing formation of Fc homodimersbetween the Fc regions of the GDF15 molecules or between Fc molecules.

In some embodiments, one or more lysine-to-aspartate mutations (K392D,K409D) are introduced into the Fc region that is joined to a GDF15region, optionally via a linker and an aspartate-to-lysine mutation(E356K) and a glutamate-to-lysine mutation (D399K) is introduced intothe Fc domain of another molecule. The aspartate residues in the Fcregion of the GDF15 molecule can associate with the lysine residues ofthe Fc molecule through electrostatic force, facilitating formation ofFc heterodimers between the Fc region of the GDF15 molecule and the Fcmolecule, and reducing or preventing formation of Fc homodimers betweenthe Fc regions of the GDF15 molecules or between Fc molecules.

In some embodiments, the GDF15 molecule comprises an Fc regioncomprising an Fc domain with a mutated hinge region. In someembodiments, the Fc domain comprises a deletion in the hinge. In someembodiments, ten amino acids from the hinge are deleted, e.g., FcΔ10. Inother embodiments, sixteen amino acids from the hinge are deleted, e.g.,FcΔ16. In some embodiments, the Fc domain comprises a hinge deletion(e.g., FcΔ10 or FcΔ16) and a charged pair mutation, such that the Fcdomain is positively or negatively charged. For example, the Fc domaincan comprise a ten-amino acid deletion in the hinge andlysine-to-aspartate mutations (K392D, K409D), such as FcΔ10(−). Inanother embodiment, the Fc domain can comprise a ten-amino acid deletionin the hinge and an aspartate-to-lysine mutation (E356K) and aglutamate-to-lysine mutation (D399K), such as an FcΔ10(+). In anotherembodiment, the Fc domain can comprise a sixteen-amino acid deletion inthe hinge and lysine-to-aspartate mutations (K392D, K409D), such asFcΔ16(−). In another embodiment, the Fc domain can comprise asixteen-amino acid deletion in the hinge and an aspartate-to-lysinemutation (E356K) and a glutamate-to-lysine mutation (D399K), such as anFcΔ16(+).

In some embodiments, an Fc molecule comprising a hinge deletion and acharged pair mutation heterodimerizes with such a GDF15 molecule. Forexample, the Fc molecule can have a hinge deletion and charged pairmutation that complements the hinge deletion and charged pair mutationof the Fc region of a GDF15 molecule. For example, an Fc molecule cancomprise an Fc domain with a ten-amino acid deletion in the hinge andlysine-to-aspartate mutations (K392D, K409D), such as FcΔ10(−), whichcan optionally comprise a C-terminal lysine (e.g., FcΔ10(−, K)). The Fcmolecule can heterodimerize with a GDF15 molecule that comprises anFcΔ10(+). In another embodiment, the Fc molecule can comprise aten-amino acid deletion in the hinge and an aspartate-to-lysine mutation(E356K) and a glutamate-to-lysine mutation (D399K), such as an FcΔ10(+),which can optionally comprise a C-terminal lysine (e.g., FcΔ10(+, K)).The Fc molecule can heterodimerize with a GDF15 molecule that comprisesan FcΔ10(−). In another embodiment, the Fc molecule can comprise asixteen-amino acid deletion in the hinge and lysine-to-aspartatemutations (K392D, K409D), such as FcΔ16(−), which can optionallycomprise a C-terminal lysine (e.g., FcΔ16(−, K)). The Fc molecule whichcan heterodimerize with a GDF15 molecule that comprises an FcΔ16(+). Inanother embodiment, the Fc molecule can comprise a sixteen-amino aciddeletion in the hinge and an aspartate-to-lysine mutation (E356K) and aglutamate-to-lysine mutation (D399K), such as an FcΔ16(+), which canoptionally comprise a C-terminal lysine (e.g., FcΔ16(−, K)). The Fcmolecule can heterodimerize with a GDF15 molecule that comprises anFcΔ16(−).

In some embodiments, the Fc region or Fc molecule comprises an Fc domainwith an L234A and/or L235A mutation, wherein 234 and 235 are thepositions using EU numbering and corresponds to the positions as notedin Tables 3-5. The Fc domain can comprise an L234A mutation, an L235Amutation, a charged pair mutation, a hinge deletion, or any combinationthereof. In some embodiments, the Fc domain comprises both an L234Amutation and an L235A mutation. In some embodiments, the Fc domaincomprises a hinge deletion, an L234A mutation, an L235A mutation, and acharged pair mutation, such as FcΔ10(+, L234A/L235A), FcΔ10(−,L234A/L235A), FcΔ16(+, L234A/L235A), or FcΔ16(−, L234A/L235A). In someembodiments, the Fc domain comprises an optional C-terminal lysine,e.g., FcΔ10(+,K,L234A/L235A), FcΔ10(−,K,L234A/L235A),FcΔ16(+,K,L234A/L235A), or FcΔ16(−,K,L234A/L235A).

In some embodiments, the Fc region or Fc molecule comprises an Fc domainwith a “cysteine clamp.” A cysteine clamp mutation involves theintroduction of a cysteine into the Fc domain at a specific locationthrough mutation so that when incubated with another Fc domain that alsohas a cysteine introduced at a specific location through mutation, adisulfide bond (cysteine clamp) may be formed between the two Fc domains(e.g., between an FcΔ16 (+) domain having a “cysteine clamp” mutationand an FcΔ16(−) domain having a “cysteine clamp” mutation). The cysteinecan be introduced into the CH3 domain of an Fc domain. In someembodiments, an Fc domain may contain one or more such cysteine clampmutations. In one embodiment, a cysteine clamp is provided byintroducing a serine to cysteine mutation (S354C, wherein 354 is theposition using EU numbering, and corresponds to the position as noted inTables 3-5) into a first Fc domain and a tyrosine to cysteine mutation(Y349C, wherein 349 is the position using EU numbering, and correspondsto the position as noted in Tables 3-5) into a second Fc domain. In oneembodiment, a GDF15 molecule comprises an Fc region comprising an Fcdomain with a cysteine clamp, a negatively charged pair mutation and asixteen-amino acid hinge deletion (e.g., GDF15- FcΔ16(−,CC)), and an Fcmolecule comprising an Fc domain comprising a cysteine clamp, apositively charged pair mutation and a sixteen-amino acid hingedeletion, and an optional C-terminal lysine (e.g., FcΔ16(+,K,CC)). Thecysteine clamp may augment the heterodimerization of the GDF-Fc moleculewith the Fc molecule.

Examples of Fc regions that can be used in a GDF15 molecule are shown inTable 3.

TABLE 3 Fc Regions SEQ ID NO: Designation Sequence 26 FcΔ10(−)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY D T TPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Underlined and bolded residues are K392D andK409D mutations. 27 FcΔ10(+) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR K EMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGUnderlined and bolded residues are E356K and D399K mutations. 28FcΔ10(−, CC) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNY D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGUnderlined and italicized residue is Y349C mutation;underlined and bolded residues are K392D and K409D mutations. 29FcΔ16(−, CC) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNY D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGUnderlined and italicized residue is Y349C mutation;underlined and bolded residues are K392D and K409D mutations. 30FcΔ16(−) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNY D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGUnderlined and bolded residues are K392D and K409D mutations. 31FcΔ10(−, L234A/L235A) APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNY D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGUnderlined and italicized residues are L234A andL235A mutations; underlined and bolded residues areK392D and K409D mutations.

Examples of Fc molecules are shown in Table 4, in which the C-terminallysine is optional.

TABLE 4 Fc Molecules SEQ ID NO: Designation Sequence 32 FcΔ10(+, K)APELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPP SR KEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVL K SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKUnderlined and bolded residues are E356K and D399K mutations. 33FcΔ10(−, K) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYD TTPPVLDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKUnderlined and bolded residues are K392D and K409D mutations. 34FcΔ10(+, K, CC) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C R K EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKUnderlined and italicized residue is S354Cmutation; underlined and bolded residues are E356K and D399K mutations.35 FcΔ16(+, K, CC) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C R K EMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKUnderlined and italicized residue is S354Cmutation; underlined and bolded residues are E356K and D399K mutations.36 FcΔ16(+, K) GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR K EMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKUnderlined and bolded residues are E356K and D399K mutations. 37FcΔ10(+, K, L234A/L235A) APE AA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SR K EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL K SDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKUnderlined and italicized residues are L234A andL235A mutations; underlined and bolded residuesare E356K and D399K mutations.

The Fc molecules can be used to dimerize with a molecule comprising acomplementary Fc domain. For example, an Fc molecule of FcΔ10(+,K) candimerize with a molecule comprising an Fc region comprising a ten-aminoacid hinge deletion and a negatively charged pair mutation such asFcΔ10(−) (e.g., a GDF15 molecule comprising an Fc region of FcΔ10(−)).An Fc molecule of FcΔ10(−,K) can dimerize with a molecule comprising anFc region comprising a ten-amino acid hinge deletion and a negativelycharged pair mutation such as FcΔ10(+) (e.g., a GDF15 moleculecomprising an Fc region of FcΔ10(+)).

An Fc molecule of FcΔ10(+,K,CC) can dimerize with a molecule comprisingan Fc region comprising a ten-amino acid hinge deletion and a negativelycharged pair mutation such as FcΔ10(−,CC) (e.g., a GDF15 moleculecomprising an Fc region of FcΔ10(−, CC)). An Fc molecule ofFcΔ16(+,K,CC) can dimerize with a molecule comprising an Fc regioncomprising a ten-amino acid hinge deletion and a negatively charged pairmutation such as FcΔ16(−, CC) (e.g., a GDF15 molecule comprising an Fcregion of FcΔ16(−, CC)). An Fc molecule of FcΔ16(+,K) can dimerize witha molecule comprising an Fc region comprising a ten-amino acid hingedeletion and a negatively charged pair mutation such as FcΔ16(−) (e.g.,a GDF15 molecule comprising an Fc region of FcΔ16(+)). An Fc molecule ofFcΔ10(+,K,L234A/L235A) can dimerize with a molecule comprising an Fcregion comprising a ten-amino acid hinge deletion and a negativelycharged pair mutation such as FcΔ10(−,L234A/L235A) (e.g., a GDF15molecule comprising an Fc region of FcΔ10(−, L234A/L235A)).

Examples of GDF15 molecules that are GDF15-Fc fusion proteins are shownin Table 5.

TABLE 5 GDF15 Molecules GDF15-Fc Fusion Protein ComponentsGDF15-Fc Fusion Protein SEQ ID NOs SEQ Fc GDF15 ID NO. DesignationSequence Region Linker Region 38 scFc7- GGGERKSSVECPPCPAPP — — — GDF15VAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSV LTVVHQDWLNGKEYKCK VSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGS GGGGSERKSSVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGSGGGGSGGGGSGGGGSGG GGSARNGDHCPLGPGRC CRLHTVRASLEDLGWAD WVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHR LKPDTVPAPCCVPASYNP MVLIQKTDTGVSLQTYDD LLAKDCHCI 39FcΔ10(−)- APELLGGPSVFLFPPKPKD  26 21 6 (G4S)4- TLMISRTPEVTCVVVDVS GDF15HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGG GSGGGGSARNGDHCPLG PGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI GACPSQFRAANMHAQIKT SLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCI Underlined and boldedresidues are K392D and K409D mutations. 40 FcΔ10(+)- APELLGGPSVFLFPPKPKD27 23 6 (G4)-GDF15 TLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR K EMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN YKTTPPVL KSDGSFFLYS KLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGGGGGARNGDHCPLGPGRCCRLHTVRASLEDL GWADWVLSPREVQVTM CIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCV PASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCIUnderlined and and bolded residues are E356K and D399K mutations. 41FcΔ10(−)- APELLGGPSVFLFPPKPKD 26 — 13 GDF15(43) TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGDHCPLGPGRCCRL HTVRASLEDLGWADWVLSPREVQVTMCIGACPSQF RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLA KDCHCI Underlined and bolded residues are K392D andK409D mutations. 42 FcΔ10(−)- APELLGGPSVFLFPPKPKD 26 — 15 GDF15(N3D)TLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGARDGDHCPLGPGRC CRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHR LKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI Underlined and boldedresidues are K392D and K409D mutations. 43 FcΔ10(−, CC)-APELLGGPSVFLFPPKPKD 28 — 13 GDF15(Δ3) TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQV C TLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGDHCPLGPGRCCRL HTVRASLEDLGWADWVLSPREVQVTMCIGACPSQF RAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLA KDCHCI Underlined and italicizedresidue is Y349C mutation; underlined and bolded residues are K392D andK409D mutations. 44 FcΔ10(−, CC)- APELLGGPSVFLFPPKPKD 28 — 15 GDF15(N3D)TLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQV C TLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGARDGDHCPLGPGRC CRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHR LKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI Underlined and italicizedresidue is Y349C mutation; underlined and bolded residues are K392D andK409D mutations. 45 FcΔ16(−, CC)- GPSVFLFPPKPKDTLMISR 29 — 17 GDF15(Δ3/DTPEVTCVVVDVSHEDPEV 5E) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP REPQV C TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNY D TTPPV LDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGEHC PLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVT MCIGACPSQFRAANMHA QIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV SLQTYDDLLAKDCHCI Underlined and italicizedresidue is Y349C mutation; underlined and bolded residues are K392D andK409D mutations. 46 FcΔ16(−, CC)- GPSVFLFPPKPKDTLMISR 29 — 18 GDF15(N3Q/TPEVTCVVVDVSHEDPEV D5E) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP REPQV C TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNY D TTPPV LDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGARQG EHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREV QVTMCIGACPSQFRAAN MHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDC HCI Underlined and italicizedresidue is Y349C mutation; underlined and bolded residues are K392D andK409D mutations. 47 FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 — 18 GDF15(N3Q/TPEVTCVVVDVSHEDPEV D5E) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNY D TTPPV LDSDGSFFLYS D LTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGARQG EHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREV QVTMCIGACPSQFRAAN MHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDC HCI Underlined and boldedresidues are K392D and K409D mutations. 48 FcΔ16(−)- GPSVFLFPPKPKDTLMISR30 25 6 (G4Q)4- TPEVTCVVVDVSHEDPEV GDF15 KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY D TTPPV LDSDGSFFLYS DLTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGG QGGGGQGGGGQGGGGQARNGDHCPLGPGRCCRL HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCIUnderlined and bolded residues are K392D and K409D mutations. 49FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 25 14 (G4Q)4- TPEVTCVVVDVSHEDPEVGDF15(N3Q) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGGQGGGGQGGGGQGGGGQ ARQGDHCPLGPGRCCRL HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCIUnderlined and bolded residues are K392D and K409D mutations. 50FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 25 18 (G4Q)4- TPEVTCVVVDVSHEDPEVGDF15(N3Q/ KFNWYVDGVEVHNAKT D5E) KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGGQGGGGQGGGGQGGGGQ ARQGEHCPLGPGRCCRLH TVRASLEDLGWADWVLS PREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPD TVPAPCCVPASYNPMVLI QKTDTGVSLQTYDDLLA KDCHCIUnderlined and bolded residues are K392D and K409D mutations. 51FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 20 14 (G4S)2- TPEVTCVVVDVSHEDPEVGDF15(N3Q) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGGSGGGGSARQGDHCPLGP GRCCRLHTVRASLEDLG WADWVLSPREVQVTMCI GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA SYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCIUnderlined and bolded residues are K392D and K409D mutations. 52FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 20 18 (G4S)2- TPEVTCVVVDVSHEDPEVGDF15(N3Q/ KFNWYVDGVEVHNAKT D5E) KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGGSGGGGSARQGEHCPLGPG RCCRLHTVRASLEDLGW ADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTS LHRLKPDTVPAPCCVPAS YNPMVLIQKTDTGVSLQT YDDLLAKDCHCIUnderlined and bolded residues are K392D and K409D mutations. 53FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 19 14 G4S- TPEVTCVVVDVSHEDPEVGDF15(N3Q) KFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGGSARQGDHCPLGPGRCCRL HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCIUnderlined and bolded residues are K392D and K409D mutations. 54FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 19 18 G4S- TPEVTCVVVDVSHEDPEVGDF15(N3Q/ KFNWYVDGVEVHNAKT D5E) KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGGGGGSARQGEHCPLGPGRCCRL HTVRASLEDLGWADWVL SPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKP DTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLA KDCHCIUnderlined and bolded residues are K392D and K409D mutations. 55FcΔ16(−)- GPSVFLFPPKPKDTLMISR 30 — 14 GDF15(N3Q) TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAV EWESNGQPENNY DTTPPV LDSDGSFFLYS D LTVDKS RWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGARQGDHCPLGPGRCCRLHTVRA SLEDLGWADWVLSPREV QVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVP APCCVPASYNPMVLIQKT DTGVSLQTYDDLLAKDC HCIUnderlined and bolded residues are K392D and K409D mutations. 56FcΔ10(−, APE AA GGPSVFLFPPKPKD 31 25 14 L234A/L235 TLMISRTPEVTCVVVDVSA)-(G4Q)4- HEDPEVKFNWYVDGVEV GDF15(N3Q) HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGGGGGQGGGGQGGG GQGGGGQARQGDHCPLGPGRCCRLHTVRASLEDLG WADWVLSPREVQVTMCI GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPA SYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCIUnderlined and italicized residues are L234A andL235A mutations; underlined and bolded residues are K392D and K409Dmutations. 57 FcΔ10(−, APE AA GGPSVFLFPPKPKD 31 25 18 L234A/L235TLMISRTPEVTCVVVDVS A)-(G4Q)4- HEDPEVKFNWYVDGVEV GDF15(N3Q/HNAKTKPREEQYNSTYR D5E) VVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENN Y DTTPPVLDSDGSFFLYS D LTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLS LSPGGGGGQGGGGQGGGGQGGGGQARQGEHCPLG PGRCCRLHTVRASLEDLG WADWVLSPREVQVTMCIGACPSQFRAANMHAQIKT SLHRLKPDTVPAPCCVPA SYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCIUnderlined and italicized residues are L234A andL235A mutations; underlined and bolded residues are K392D and K409Dmutations.

In some embodiments, the fusion protein is an scFc-GDF15 in which theGDF15 region is joined to two Fc regions. In some embodiments, thefusion protein comprises an amino acid sequence that has at least 85%,90%, 95% or 99% sequence identity to SEQ ID NO: 38. In some embodiments,the fusion protein comprises an amino acid sequence of SEQ ID NO: 38. Incalculating percent sequence identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Acomputer program that can be used to determine percent identity is theGCG program package, which includes GAP (Devereux et al., (1984) Nucl.Acid Res. 12:387; Genetics Computer Group, University of Wisconsin,Madison, Wis.). The computer algorithm GAP can be used to align the twopolypeptides or polynucleotides for which the percent sequence identityis to be determined. The sequences are aligned for optimal matching oftheir respective amino acid or nucleotide (the “matched span”, asdetermined by the algorithm). A gap opening penalty (which is calculatedas 3× the average diagonal, wherein the “average diagonal” is theaverage of the diagonal of the comparison matrix being used; the“diagonal” is the score or number assigned to each perfect amino acidmatch by the particular comparison matrix) and a gap extension penalty(which is usually 1/10 times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. In certain embodiments, a standard comparison matrix(see, Dayhoff et al., (1978) Atlas of Protein Sequence and Structure5:345-352 for the PAM 250 comparison matrix; Henikoff et al., (1992)Proc. Natl. Acad. Sci. U.S.A. 9:10915-10919 for the BLOSUM 62 comparisonmatrix) is also used by the algorithm. Parameters that can be used fordetermining percent identity using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;

Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4

Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences canresult in matching of only a short region of the two sequences, and thissmall aligned region can have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (e.g., the GAPprogram) can be adjusted if so desired to result in an alignment thatspans at least 50 contiguous amino acids of the target polypeptide.

In some embodiments, the GDF15 molecule is FcΔ10(−)-(G4S)4-GDF15,FcΔ10(+)-(G4)-GDF15, FcΔ10(−)-GDF15(Δ3), FcΔ10(−)-GDF15(N3D),FcΔ10(−,CC)-GDF15(Δ3), FcΔ10(−,CC)-GDF15(N3D),FcΔ16(−,CC)-GDF15(Δ3/D5E), FcΔ16(−,CC)-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q/D5E), FcΔ16(−)-(G4Q)4-GDF15,FcΔ16(−)-(G4Q)4-GDF15(N3Q), FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E),FcΔ16(−)-(G4S)2-GDF15(N3Q), FcΔ16(−)-(G4S)2-GDF15(N3Q/D5E),FcΔ16(−)-G4S-GDF15(N3Q), FcΔ16(−) -G4S-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q), FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q), orFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E).

In some embodiments, the GDF15 molecule comprises the amino acidsequence of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, or 57. In some embodiments, the GDF15 moleculecomprises an amino acid sequence that has 80-99%, 85%-99%, 90-99%, or95-99% sequence identity to SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, or 57. In some embodiments, theGDF15 molecule comprises an amino acid sequence that has at least 85%sequence identity to SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, or 57. In some embodiments, the GDF15molecule comprises an amino acid sequence that has at least 90% sequenceidentity to SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, or 57. In some embodiments, the GDF15 moleculecomprises an amino acid sequence that has at least 95% sequence identityto SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, or 57. In some embodiments, the GDF15 molecule comprisesan amino acid sequence that has at least 99% sequence identity to SEQ IDNO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, or 57.

In some embodiments, the GDF15 molecule is a FcΔ10(−)-(G4S)4-GDF15,FcΔ10(+)-(G4)-GDF15, FcΔ10(−)-GDF15(Δ3), FcΔ10(−)-GDF15(N3D),FcΔ10(−,CC)-GDF15(Δ3), FcΔ10(−,CC)-GDF15(N3D),FcΔ16(−,CC)-GDF15(Δ3/D5E), FcΔ16(−,CC)-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q/D5E), FcΔ16(−)-(G4Q)4-GDF15,FcΔ16(−)-(G4Q)4-GDF15(N3Q), FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E),FcΔ16(−)-(G4S)2-GDF15(N3Q), FcΔ16(−)-(G4S)2-GDF15(N3Q/D5E),FcΔ16(−)-G4S-GDF15(N3Q), FcΔ16(−) -G4S-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q), FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q), orFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule. In someembodiments, the GDF15 molecule is a FcΔ10(−)-(G4S)4-GDF15,FcΔ10(+)-(G4)-GDF15, FcΔ10(−)-GDF15(Δ3), FcΔ10(−)-GDF15(N3D),FcΔ10(−,CC)-GDF15(Δ3), FcΔ10(−,CC)-GDF15(N3D),FcΔ16(−,CC)-GDF15(Δ3/D5E), FcΔ16(−,CC)-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q/D5E), FcΔ16(−)-(G4Q)4-GDF15,FcΔ16(−)-(G4Q)4-GDF15(N3Q), FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E),FcΔ16(−)-(G4S)2-GDF15(N3Q), FcΔ16(−)-(G4S)2-GDF15(N3Q/D5E),FcΔ16(−)-G4S-GDF15(N3Q), FcΔ16(−)-G4S-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q), FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q), orFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule that has 80-99%,85%-99%, 90-99%, or 95-99% sequence identity to its Fc region and/orGDF15 region.

In some embodiments, the GDF15 molecule is a FcΔ10(−)-(G4S)4-GDF15,FcΔ10(+)-(G4)-GDF15, FcΔ10(−)-GDF15(Δ3), FcΔ10(−)-GDF15(N3D),FcΔ10(−,CC)-GDF15(Δ3), FcΔ10(−,CC)-GDF15(N3D),FcΔ16(−,CC)-GDF15(Δ3/D5E), FcΔ16(−,CC)-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q/D5E), FcΔ16(−)-(G4Q)4-GDF15,FcΔ16(−)-(G4Q)4-GDF15(N3Q), FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E),FcΔ16(−)-(G4S)2-GDF15(N3Q), FcΔ16(−)-(G4S)2-GDF15(N3Q/D5E),FcΔ16(−)-G4S-GDF15(N3Q), FcΔ16(−) -G4S-GDF15(N3Q/D5E),FcΔ16(−)-GDF15(N3Q), FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q), orFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) molecule that has at least85%, 90%, 95% or 99% sequence identity to its Fc region and/or GDF15region. For example, a FcΔ10(−)-(G4S)4-GDF15 molecule with 80-99%,85%-99%, 90-99%, or 95-99% sequence identity to its Fc region and/orGDF15 region, includes a GDF15 molecule with an Fc region that has aten-amino acid deletion of the hinge region and a negatively chargedpair mutation, and has 80-99%, 85%-99%, 90-99%, or 95-99% sequenceidentity to SEQ ID NO: 26 and/or a GDF15 region that has 80-99%,85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO: 6. Forexample, a FcΔ10(−)-(G4S)4-GDF15 molecule with at least 85%, 90%, 95% or99% sequence identity to its Fc region and/or GDF15 region, includes aGDF15 molecule with an Fc region that has a ten-amino acid deletion ofthe hinge region and a negatively charged pair mutation, and has atleast 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 26 and/or aGDF15 region that has at least 85%, 90%, 95% or 99% sequence identity toSEQ ID NO: 6.

In another example, a FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) molecule with80-99%, 85%-99%, 90-99%, or 95-99% sequence identity to its Fc regionand/or a GDF15 region, includes a GDF15 molecule with an Fc region thathas a sixteen-amino acid deletion of the hinge region and a negativelycharged pair mutation that has 80-99%, 85%-99%, 90-99%, or 95-99%sequence identity to SEQ ID NO: 30 and/or a GDF15 region that has80-99%, 85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO: 18.In another example, a FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) molecule with atleast 85%, 90%, 95% or 99% sequence identity to its Fc region and/or aGDF15 region, includes a GDF15 molecule with an Fc region that has asixteen-amino acid deletion of the hinge region and a negatively chargedpair mutation that has at least 85%, 90%, 95% or 99% sequence identityto SEQ ID NO: 30 and/or a GDF15 region that has at least 85%, 90%, 95%or 99% sequence identity to SEQ ID NO: 18.

In yet another example, a FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E)molecule with 80-99%, 85%-99%, 90-99%, or 95-99% sequence identity toits Fc region and/or a GDF15 region, includes a GDF15 molecule with anFc region that has a ten-amino acid deletion of the hinge region, anegatively charged pair mutation and leucine to alanine mutations atpositions 234 and 235 and has 80-99%, 85%-99%, 90-99%, or 95-99%sequence identity to SEQ ID NO: 31 and/or a GDF15 region that has80-99%, 85%-99%, 90-99%, or 95-99% sequence identity to SEQ ID NO: 18.In yet another example, a FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E)molecule with at least 85%, 90%, 95% or 99% sequence identity to its Fcregion and/or a GDF15 region, includes a GDF15 molecule with an Fcregion that has a ten-amino acid deletion of the hinge region, anegatively charged pair mutation and leucine to alanine mutations atpositions 234 and 235 and has at least 85%, 90%, 95% or 99% sequenceidentity to SEQ ID NO: 31 and/or a GDF15 region that has at least 85%,90%, 95% or 99% sequence identity to SEQ ID NO: 18.

Also provided herein are dimers and tetramers comprising a GDF15molecule provided herein. In one embodiment, the dimer comprises aGDF15-Fc fusion comprising the amino acid sequence of any one of SEQ IDNOs: 39-57. In some embodiments, a GDF15-Fc fusion comprising the aminoacid sequence of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56 or 57 dimerizes with an Fc moleculecomprising the amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, or37 (in which the C-terminal lysine is optional), such as shown in Table6. For example, in some embodiments, the dimer is FcΔ10(−)-(G4S)4-GDF15:FcΔ10(+,K). In another embodiment, the dimer isFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q): FcΔ10(+,K,L234A/L235A). In yetanother embodiment, the dimer isFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q):FcΔ10(+,K,L234A/L235A).

TABLE 6 Dimers GDF15- Fc Fusion SEQ ID GDF15-Fc Fusion Fc Molecule SEQCorresponding Fc Molecule NO. Designation ID NO. Designation 39FcΔ10(−)-(G4S)4-GDF15 32 FcΔ10(+, K) 40 FcΔ10(+)-(G4)-GDF15 33FcΔ10(−, K) 41 FcΔ10(−)-GDF15(Δ3) 32 FcΔ10(+, K) 42 FcΔ10(−)-GDF15(N3D)32 FcΔ10(+, K) 43 FcΔ10(−, CC)-GDF15(Δ3) 34 FcΔ10(+, K, CC) 44FcΔ10(−, CC)-GDF15(N3D) 34 FcΔ10(+, K, CC) 45 FcΔ16(−, CC)-GDF15(Δ3/D5E)35 FcΔ16(+, K, CC) 46 FcΔ16(−, CC)- 35 FcΔ16(+, K, CC) GDF15(N3Q/D5E) 47FcΔ16(−)-GDF15(N3Q/D5E) 36 FcΔ16(+, K) 48 FcΔ16(−)-(G4Q)4-GDF15 36FcΔ16(+, K) 49 FcΔ16(−)-(G4Q)4- 36 FcΔ16(+, K) GDF15(N3Q) 50FcΔ16(−)-(G4Q)4- 36 FcΔ16(+, K) GDF15(N3Q/D5E) 51 FcΔ16(−)-(G4S)2- 36FcΔ16(+, K) GDF15(N3Q) 52 FcΔ16(−)-(G4S)2- 36 FcΔ16(+, K) GDF15(N3Q/D5E)53 FcΔ16(−)-G4S-GDF15(N3Q) 36 FcΔ16(+, K) 54 FcΔ16(−)-G4S- 36FcΔ16(+, K) GDF15(N3Q/D5E) 55 FcΔ16(−)-GDF15(N3Q) 36 FcΔ16(+, K) 56FcΔ10(−, L234A/L235A)- 37 FcΔ10(+, K, L234A/L235A) (G4Q)4-GDF15(N3Q) 57FcΔ10(−, L234A/L235A)- 37 FcΔ10(+, K, L234A/L235A)(G4Q)4- GDF15(N3Q/D5E)

In one embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 39 dimerizes with an Fc molecule comprising SEQ ID NO: 32(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 40 dimerizes with an Fcmolecule comprising SEQ ID NO: 33 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 41 dimerizes with an Fc molecule comprising SEQ ID NO: 32(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 42 dimerizes with an Fcmolecule comprising SEQ ID NO: 32 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 43 dimerizes with an Fc molecule comprising SEQ ID NO: 34(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 44 dimerizes with an Fcmolecule comprising SEQ ID NO: 34 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 44 dimerizes with an Fc molecule comprising SEQ ID NO: 34(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 45 dimerizes with an Fcmolecule comprising SEQ ID NO: 35 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 46 dimerizes with an Fc molecule comprising SEQ ID NO: 35(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 47 dimerizes with an Fcmolecule comprising SEQ ID NO: 36 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 48 dimerizes with an Fc molecule comprising SEQ ID NO: 36(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 49 dimerizes with an Fcmolecule comprising SEQ ID NO: 36 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 50 dimerizes with an Fc molecule comprising SEQ ID NO: 36(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 51 dimerizes with an Fcmolecule comprising SEQ ID NO: 36 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 52 dimerizes with an Fc molecule comprising SEQ ID NO: 36(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 53 dimerizes with an Fcmolecule comprising SEQ ID NO: 36 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 54 dimerizes with an Fc molecule comprising SEQ ID NO: 36(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 55 dimerizes with an Fcmolecule comprising SEQ ID NO: 36 (C-terminal lysine optional). Inanother embodiment, a GDF15-Fc fusion comprising the amino acid sequenceof SEQ ID NO: 56 dimerizes with an Fc molecule comprising SEQ ID NO: 37(C-terminal lysine optional). In another embodiment, a GDF15-Fc fusioncomprising the amino acid sequence of SEQ ID NO: 57 dimerizes with an Fcmolecule comprising SEQ ID NO: 37 (C-terminal lysine optional).

In some embodiments, the dimers form tetramers. For example, the dimersin Table 6 can form tetramers. In some embodiments, the tetramers areformed from the same dimers. In some embodiments, two dimers ofFcΔ10(−)-(G4S)4-GDF15:FcΔ10(+,K); FcΔ10(+)-(G4)-GDF15:FcΔ10(−,K);FcΔ10(−)-GDF15(Δ3):FcΔ10(+,K); FcΔ10(−)-GDF15(N3D):FcΔ10(+,K);FcΔ10(−,CC)-GDF15(Δ3):FcΔ10(+,K,CC);FcΔ10(−,CC)-GDF15(N3D):FcΔ10(+,K,CC);FcΔ16(−,CC)-GDF15(Δ3/D5E):FcΔ16(+,K,CC);FcΔ16(−,CC)-GDF15(N3Q/D5E):FcΔ16(+,K,CC);FcΔ16(−)-GDF15(N3Q/D5E):FcΔ16(+,K); FcΔ16(−)-(G4Q)4-GDF15:FcΔ16(+,K);FcΔ16(−)-(G4Q)4-GDF15(N3Q):FcΔ16(+,K);FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E):FcΔ16(+,K);FcΔ16(−)-(G45)2-GDF15(N3Q):FcΔ16(+,K);FcΔ16(−)-(G4S)2-GDF15(N3Q/D5E):FcΔ16(+,K);FcΔ16(−)-G4S-GDF15(N3Q):FcΔ16(+,K); FcΔ16(−)-G4S-GDF15(N3Q/D5E):FcΔ16(+,K); FcΔ16(−)-GDF15(N3Q): FcΔ16(+,K);FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q):FcΔ10(+,K,L234A/L235A); orFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):FcΔ10(+,K,L234A/L235A) form atetramer, such as through the dimerization of the two GDF15 regions.

Also provided herein are host cells comprising the nucleic acids andvectors for producing the GDF15 and Fc molecules disclosed herein. Invarious embodiments, the vector or nucleic acid is integrated into thehost cell genome, which in other embodiments the vector or nucleic acidis extra-chromosomal.

Recombinant cells, such as yeast, bacterial (e.g., E. coli), andmammalian cells (e.g., immortalized mammalian cells) comprising such anucleic acid, vector, or combinations of either or both thereof areprovided. In various embodiments, cells comprising a non-integratednucleic acid, such as a plasmid, cosmid, phagemid, or linear expressionelement, which comprises a sequence coding for expression of a GDF15molecule and/or an Fc molecule. In some embodiments, the cell comprisesa nucleic acid for producing a GDF15 molecule and another cell comprisesa nucleic acid for producing an Fc molecule for dimerization with theGDF15 molecule (e.g., a vector for encoding a GDF15 molecule in one celland a second vector for encoding an Fc molecule in a second cell). Inother embodiments, a host cell comprises a nucleic acid for producing aGDF15 molecule and an Fc molecule (e.g., a vector that encodes bothmolecules). In another embodiment, a host cell comprises a nucleic acidfor producing a GDF15 molecule and another nucleic acid for producing anFc molecule (e.g., two separate vectors, one that encodes a GDF15molecule and one that encodes an Fc molecule, in a single host cell).

A vector comprising a nucleic acid sequence encoding a GDF15 moleculeand/or an Fc molecule can be introduced into a host cell bytransformation or by transfection, such as by methods known in the art.

A nucleic acid encoding a GDF15 molecule can be positioned in and/ordelivered to a host cell or host animal via a viral vector. A viralvector can comprise any number of viral polynucleotides, alone or incombination with one or more viral proteins, which facilitate delivery,replication, and/or expression of the nucleic acid of the invention in adesired host cell. The viral vector can be a polynucleotide comprisingall or part of a viral genome, a viral protein/nucleic acid conjugate, avirus-like particle (VLP), or an intact virus particle comprising viralnucleic acids and a nucleic acid encoding a polypeptide comprising aGDF15 region. A viral particle viral vector can comprise a wild-typeviral particle or a modified viral particle. The viral vector can be avector which requires the presence of another vector or wild-type virusfor replication and/or expression (e.g., a viral vector can be ahelper-dependent virus), such as an adenoviral vector amplicon. Suitableviral vector particles in this respect, include, for example, adenoviralvector particles (including any virus of or derived from a virus of theadenoviridae), adeno-associated viral vector particles (AAV vectorparticles) or other parvoviruses and parvoviral vector particles,papillomaviral vector particles, flaviviral vectors, alphaviral vectors,herpes viral vectors, pox virus vectors, retroviral vectors, includinglentiviral vectors.

A GDF15 molecule can be isolated using standard protein purificationmethods. A polypeptide comprising a GDF15 region can be isolated from acell that has been engineered to express a polypeptide comprising aGDF15 region, for example a cell that does not naturally express nativeGDF15. Protein purification methods known in the art can be employed toisolate GDF15 molecules, as well as associated materials and reagents.Methods of purifying a GDF15 molecule are also provided in the Examplesherein. Additional purification methods that may be useful for isolatingGDF15 molecules can be found in references such as Bootcov M R, 1997,Proc. Natl. Acad. Sci. USA 94:11514-9, Fairlie W D, 2000, Gene 254:67-76.

Pharmaceutical compositions comprising a GDF15 molecule (and optionally,an Fc molecule, such as a dimer or tetramer disclosed herein) are alsoprovided. Such polypeptide pharmaceutical compositions can comprise atherapeutically effective amount of a GDF15 molecule in admixture with apharmaceutically or physiologically acceptable formulation agent orcarrier selected for suitability with the mode of administration. Thepharmaceutically or physiologically acceptable formulation agent can beone or more formulation agents suitable for accomplishing or enhancingthe delivery of a GDF15 molecule into the body of a human or non-humansubject. Pharmaceutically acceptable substances such as wetting oremulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the GDF15 molecule can also act as, or form acomponent of, a formulation carrier. Acceptable pharmaceuticallyacceptable carriers are preferably nontoxic to recipients at the dosagesand concentrations employed. The pharmaceutical composition can containformulation agent(s) for modifying, maintaining, or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorption,or penetration of the composition.

The effective amount of pharmaceutical composition comprising a GDF15molecule which is to be employed therapeutically will depend, forexample, upon the therapeutic context and objectives. One skilled in theart will appreciate that the appropriate dosage levels for treatmentwill thus vary depending, in part, upon the molecule delivered, theindication for which a GDF15 molecule is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the subject. The frequencyof dosing will depend upon the pharmacokinetic parameters of the GDF15molecule in the formulation being used.

The route of administration of the pharmaceutical composition can beorally; through injection by intravenous, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, intraportal, or intralesional routes; by sustainedrelease systems (which may also be injected); or by implantationdevices. Where desired, the compositions can be administered by bolusinjection or continuously by infusion, or by an implantation device. Thecomposition can also be administered locally via implantation of amembrane, sponge, or other appropriate material onto which the desiredmolecule has been absorbed or encapsulated. Where an implantation deviceis used, the device can be implanted into any suitable tissue or organ,and delivery of the desired molecule can be via diffusion, timed-releasebolus, or continuous administration.

A GDF15 molecule can be used to treat, diagnose or ameliorate, ametabolic condition or disorder. In one embodiment, the metabolicdisorder is diabetes, e.g., type 2 diabetes. In another embodiment, themetabolic condition or disorder is obesity. In other embodiments, themetabolic condition or disorder is dyslipidemia, elevated glucoselevels, elevated insulin levels or diabetic nephropathy. For example, ametabolic condition or disorder that can be treated or ameliorated usinga GDF15 molecule includes a state in which a human subject has a fastingblood glucose level of 125 mg/dL or greater, for example 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or greaterthan 200 mg/dL. Blood glucose levels can be determined in the fed orfasted state, or at random. The metabolic condition or disorder can alsocomprise a condition in which a subject is at increased risk ofdeveloping a metabolic condition. For a human subject, such conditionsinclude a fasting blood glucose level of 100 mg/dL. Conditions that canbe treated using a pharmaceutical composition comprising a GDF15molecule can also be found in the American Diabetes AssociationStandards of Medical Care in Diabetes Care 2011, American DiabetesAssociation, Diabetes Care Vol. 34, No. Supplement 1, S11-S61, 2010.

The administration can be performed such as by intravenous (IV)injection, intraperitoneal (IP) injection, subcutaneous injection,intramuscular injection, or orally in the form of a tablet or liquidformation. A therapeutically effective dose of a GDF15 molecule willdepend upon the administration schedule, the unit dose of agentadministered, whether the GDF15 molecule is administered in combinationwith other therapeutic agents, the immune status and the health of therecipient. A therapeutically effective dose is an amount of a GDF15molecule that elicits a biological or medicinal response in a tissuesystem, animal, or human being sought by a researcher, medical doctor,or other clinician, which includes alleviation or amelioration of thesymptoms of the disease or disorder being treated, i.e., an amount of aGDF15 molecule that supports an observable level of one or more desiredbiological or medicinal response, for example, lowering blood glucose,insulin, triglyceride, or cholesterol levels; reducing body weight;improving glucose tolerance, energy expenditure, or insulin sensitivity;or reducing food intake. A therapeutically effective dose of a GDF15molecule can also vary with the desired result.

Also provided herein is a method comprising measuring a baseline levelof one or more metabolically-relevant compounds such as glucose,insulin, cholesterol, lipid in a subject, administering a pharmaceuticalcomposition comprising a GDF15 molecule to the subject, and after adesired period of time, measure the level of the one or moremetabolically-relevant compounds (e.g., blood glucose, insulin,cholesterol, lipid) in the subject. The two levels can then be comparedto determine the relative change in the metabolically-relevant compoundin the subject. Depending on the outcome of that comparison another doseof the pharmaceutical composition can be administered to achieve adesired level of one or more metabolically-relevant compound.

A pharmaceutical composition comprising a GDF15 molecule can beco-administered with another compound or therapeutic agent. A GDF15molecule (and optionally, its corresponding Fc molecule) can beadministered in combination with another therapeutic agent, such as anagent that lowers blood glucose, insulin, triglyceride, or cholesterollevels; lowers body weight; reduces food intake; improves glucosetolerance, energy expenditure, or insulin sensitivity; or anycombination thereof (e.g., antidiabetic agent, hypolipidemic agent,anti-obesity agent, anti-hypertensive agent, or agonist of peroxisomeproliferator-activator receptor). The identity and properties of acompound co-administered with the GDF15 molecule will depend on thenature of the condition to be treated or ameliorated. The agentadministered with a GDF15 molecule disclosed herein can be a GLP-1Ragonist, such as GLP-1 or an analog thereof; or an exendin, exendinanalog, or exendin agonist. A non-limiting list of examples of compoundsthat can be administered in combination with the pharmaceuticalcomposition include liraglutide, rosiglitizone, pioglitizone,repaglinide, nateglitinide, metformin, exenatide, stiagliptin,pramlintide, glipizide, glimeprirideacarbose, orlistat, lorcaserin,phenterminetopiramate, naltrexonebupropion, setmelanotide, semaglutide,efpeglenatide, canagliflozin, LIK-066, SAR-425899, Tt-401, FGFR4Rx,HDV-biotin and miglitol.

In one embodiment, a GDF15 molecule comprising the amino acid sequenceof SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56 or 57 is administered with another compound ortherapeutic agent, such as liraglutide.

In another embodiment, a GDF15 molecule and corresponding Fc moleculecomprising the amino acid sequences of SEQ ID NOs: 39 and 32 (C-terminallysine optional), respectively; SEQ ID NOs: 40 and 33 (C-terminal lysineoptional), SEQ ID NOs: 41 and 32 (C-terminal lysine optional),respectively; SEQ ID NOs: 42 and 32 (C-terminal lysine optional),respectively; SEQ ID NOs: 43 and 34 (C-terminal lysine optional),respectively; SEQ ID NOs: 44 and 34 (C-terminal lysine optional),respectively; SEQ ID NOs: 45 and 35 (C-terminal lysine optional),respectively; SEQ ID NOs: 46 and 35 (C-terminal lysine optional),respectively; SEQ ID NOs: 47 and 36 (C-terminal lysine optional)respectively; SEQ ID NOs: 48 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 49 and 36 (C-terminal lysine optional)respectively; SEQ ID NOs: 50 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 51 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 52 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 53 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 54 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 55 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 56 and 37 (C-terminal lysine optional),respectively; or SEQ ID NOs: 57 and 37 (C-terminal lysine optional),respectively; is administered with another compound or therapeuticagent, such as liraglutide.

In another embodiment, a GDF15 molecule and corresponding Fc moleculecomprising the amino acid sequences of SEQ ID NOs: 50 and 36 (C-terminallysine optional), respectively, is administered with another compound ortherapeutic agent, such as liraglutide. In another embodiment, a GDF15molecule and corresponding Fc molecule comprising the amino acidsequences of SEQ ID NOs: 57 and 37 (C-terminal lysine optional),respectively, is administered with another compound or therapeuticagent, such as liraglutide.

A GDF15 molecule administered with another therapeutic agent can includeconcurrent administration of a therapeutically effective amount of theGDF15 molecule (and optionally, its corresponding Fc molecule) and atherapeutically effective amount of the other therapeutic agent. A GDF15molecule administered with another therapeutic agent can includesubsequent administration of a therapeutically effective amount of theGDF15 molecule (and optionally, its corresponding Fc molecule) and atherapeutically effective amount of the other therapeutic agent, e.g.,administration of a therapeutically effective amount of the GDF15molecule (and optionally, its corresponding Fc molecule) followed by atherapeutically effective amount of the other therapeutic agent oradministration of a therapeutically effective amount of the othertherapeutic agent followed by administration of a therapeuticallyeffective amount of the GDF15 molecule (and optionally, itscorresponding Fc molecule). Administration of a therapeuticallyeffective amount of the GDF15 molecule (and optionally, itscorresponding Fc molecule) can be at least 1, 2, 3, 4, 5, 6, or 7 daysafter administration of a therapeutically effective amount of the othertherapeutic agent. In another embodiment, administration of atherapeutically effective amount of a therapeutically effective amountof the other therapeutic agent can be at least 1, 2, 3, 4, 5, 6, or 7days after at least 1, 2, 3, 4, 5, 6, or 7 days after administration ofa therapeutically effective amount of the GDF15 molecule (andoptionally, its corresponding Fc molecule).

A GDF15 molecule administered concurrently with another therapeuticagent can comprise administration of a composition comprising both theGDF15 molecule (and optionally its corresponding Fc molecule) and theother therapeutic agent, e.g., a therapeutically effective amount of theGDF15 molecule (and optionally its corresponding Fc molecule) iscombined with a therapeutically effective amount of the other agentprior to administration. In another embodiment, concurrentadministration of GDF15 molecule (and optionally its corresponding Fcmolecule) and another therapeutic agent can comprise concurrentadministration of a first composition comprising the GDF15 molecule anda second composition comprising the other therapeutic agent.

In some embodiments, administration of a GDF15 molecule with anothertherapeutic agent has a synergistic effect. In one embodiment, theeffect is greater than the GDF15 molecule (and optionally itscorresponding Fc molecule) alone or the other agent. In anotherembodiment, the effect is greater than an additive effect of both agents(the GDF15 molecule, and optionally its corresponding Fc molecule, plusthe other agent). In one embodiment, combination therapy (i e,administration of a GDF15 molecule, optionally with its corresponding Fcmolecule, with another therapeutic agent) has a greater than 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 fold effectthan GDF15 monotherapy (administration of the GDF15 molecule, andoptionally its corresponding Fc molecule). In another embodiment,combination therapy (i.e., administration of a GDF15 molecule,optionally with its corresponding Fc molecule, with another therapeuticagent) has a greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 fold effect than monotherapy with the otheragent. The effect can be the amount of body weight lost (e.g., thedecrease in total mass or percent body change); the decrease in bloodglucose, insulin, triglyceride, or cholesterol levels; the improvementin glucose tolerance, energy expenditure, or insulin sensitivity; or thereduction food intake. The synergistic effect can be about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49, 56, 63, or 70days after administration.

In one embodiment, a GDF15 molecule and corresponding Fc moleculecomprising the amino acid sequences of SEQ ID NOs: 39 and 32 (C-terminallysine optional), respectively; SEQ ID NOs: 40 and 33 (C-terminal lysineoptional), SEQ ID NOs: 41 and 32 (C-terminal lysine optional),respectively; SEQ ID NOs: 42 and 32 (C-terminal lysine optional),respectively; SEQ ID NOs: 43 and 34 (C-terminal lysine optional),respectively; SEQ ID NOs: 44 and 34 (C-terminal lysine optional),respectively; SEQ ID NOs: 45 and 35 (C-terminal lysine optional),respectively; SEQ ID NOs: 46 and 35 (C-terminal lysine optional),respectively; SEQ ID NOs: 47 and 36 (C-terminal lysine optional)respectively; SEQ ID NOs: 48 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 49 and 36 (C-terminal lysine optional)respectively; SEQ ID NOs: 50 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 51 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 52 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 53 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 54 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 55 and 36 (C-terminal lysine optional),respectively; SEQ ID NOs: 56 and 37 (C-terminal lysine optional),respectively; or SEQ ID NOs: 57 and 37 (C-terminal lysine optional),respectively; administered with a GLP-1R agonist (e.g., liraglutide orexendin, or an analog or agonist thereof) has a greater than 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 fold effectthan GDF15 monotherapy; a greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25,26, 27, 28, 29, or 30 fold effect than GLP-1R agonistmonotherapy (i.e., administration of GLP-1R agonist alone); or both,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49,56, 63, or 70 days after administration of the agent(s).

The detailed description and following examples illustrate the presentinvention and are not to be construed as limiting the present inventionthereto. Various changes and modifications can be made by those skilledin the art on the basis of the description of the invention, and suchchanges and modifications are also included in the present invention.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved, are provided for illustrative purposes only and are not to beconstrued as limiting the present invention.

Example 1: GDF15(WT)-Linker-Fc Molecules

The GDF15 molecules of scFc-GDF15 (SEQ ID NO: 38) andFcΔ10(−)-(G45)4-GDF15 (SEQ ID NO: 39) were produced and the activity ofthe molecules tested.

FcΔ10(−)-(G4S)4-GDF15 (SEQ ID NO: 39) was stably expressed in a serumfree, suspension adapted CHO-K1 cell line. It was cloned into a stableexpression vector containing puromycin resistance while the Fc chain forforming a heterodimer with FcΔ10(−)-(G4S)4-GDF15, FcΔ10(+,K) (SEQ ID NO:32), was cloned into a hygromycin containing expression vector (Selexis,Inc.). The plasmids were transfected at a 1:1 ratio using lipofectamineLTX and cells were selected 2 days post transfection in a proprietarygrowth media containing 10 ug/mL puromycin and 600 ug/mL hygromycin.Media was exchanged 2 times per week during selection. When cellsreached about 90% viability, they were scaled up for a batch productionrun. Cells were seeded at 2×10⁶/mL in production media. The conditionedmedium (CM) produced by the cells was harvested on day 7 and clarified.Endpoint viabilities typically were above 90%.

FcΔ10(−)-(G4S)4-GDF15 (SEQ ID NO: 39) (and any paired Fc) wereclarified. Conditioned media was purified using a two-stepchromatography procedure. Approximately 5 L of the CM was applieddirectly to a GE Mab Select SuRe column that had previously beenequilibrated with Dulbecco's Phosphate Buffered Saline (PBS). The boundprotein underwent three wash steps: first, 3 column volumes (CV) of PBS;next, 1 CV of 20 mM Tris, 100 mM sodium chloride, pH 7.4; and finally, 3CV of 500 mM L-arginine, pH 7.5. These wash steps remove unbound orlightly bound media components and host cell impurities. The column wasthen re-equilibrated with 5 CV of 20 mM Tris, 100 mM sodium chloride atpH 7.4 which brought the UV absorbance back to baseline. The desiredprotein was eluted with 100 mM acetic acid at pH 3.6 and collected inbulk. The protein pool was quickly titrated to within a pH range of 5.0to 5.5 with 1 M Tris-HC1, pH 9.2. The pH adjusted protein pool was nextloaded onto a GE SP Sepharose® HP column that had been previouslyequilibrated with 20 mM 2-ethanesulfonic acid (MES) at pH 6.0. The boundprotein was then washed with 5 CV of equilibration buffer, and finallyeluted over a 20 CV, 0 to 50% linear gradient from 0 to 400 mM sodiumchloride in 20 mM MES at pH 6.0. Fractions were collected during theelution and analyzed by analytical size-exclusion chromatography(Superdex® 200) to determine the appropriate fractions to pool for ahomogeneous product. The SP HP chromatography removes product-relatedimpurities such as free Fc, clipped species, and Fc-GDF15 multimers. TheSP HP pool was then buffer exchanged into 10 mM sodium acetate, 5%proline, pH 5.2 by dialysis. It was concentrated to approximately 15mg/ml using the Sartorius Vivaspin® 20 ten kilo-dalton molecular weightcut-off centrifugal device. Finally, it was sterile filtered and theresulting solution containing the purified Fc-GDF15 molecules was storedat 5° C. Final products were assessed for identity and purity using massspectral analysis, sodium dodecyl sulfate polyacrylamide electrophoresisand size exclusion high performance liquid chromatography.

ScFc-GDF15 (SEQ ID NO: 38) was produced in a similar manner. This GDF15molecule was stably expressed in a CHO/CS9 cell line. The molecules werecloned into a stable expression vector. The plasmids (linearized) weretransfected at a 1:1 ratio using electroporation and cells were selected2 days post transfection. Media was exchanged 3 times per week duringselection. When cells reached about 90% viability, they were scaled upfor a fed batch production run. Cells were seeded at 1×10⁶/mL inproduction media and fed once when the cell number reached to4-5×10⁶/ml. The conditioned medium (CM) produced by the cells washarvested on day 10 and clarified. Endpoint viabilities typically wereabove 90%. ScFc-GDF15 was clarified and conditioned media was purifiedusing a two-step coupled chromatography procedure. Conditioned mediafrom multiple harvests were pooled and concentrated nearly 5 fold byultrafiltration using a 1 sq ft Pellicon® 2 10 kD regenerated cellulosemembrane (Millipore) by tangential flow filtration. Approximately 5 L ofthe concentrated CM was applied directly to a GE MabSelect SuRe columnthat had previously been equilibrated with Dulbecco's Phosphate BufferedSaline (PBS). The non-specifically bound protein was removed by a 12CVPBS wash step. The desired protein was eluted with 0.5% acetic acid atpH 3.5, 150 mM NaCl in 3 CV and collected in a storage loop. Thecollected protein pool was directly loaded onto a GE HiLoad 26/60Superdex 200 Prep Grade sizing column that had been previouslyequilibrated with 30 mM acetate at pH 5.0, 150 mM NaCl. Peak fractionscollected during the sizing run were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to determine the appropriatefractions to pool for a homogeneous product. The pH of the finalsizing-pool was adjusted to pH 4.5, with the addition of 10% glacialacetic acid and then buffer exchanged into 10 mM sodium acetate, 9%(w/v) sucrose, pH 4.5 by dialysis. It was concentrated to above 15 mg/mlusing a ten kilo-dalton molecular weight cut-off centrifugal device.Protein stability to freezing was tested by 3 cycles of freezing andthawing. Finally, the final lot was sterile filtered and the resultingsolution containing the purified GDF15 molecules was stored at −80° C.Final products were assessed for identity and purity using mass spectralanalysis, n-terminal sequencing, sodium dodecyl sulfate polyacrylamideelectrophoresis and size exclusion high performance liquidchromatography.

Activity of scFc-GDF15 and FcΔ10(−)-(G4S)4-GDF15 was then analyzed forin vivo activity. Cynomologus monkeys (n =10 per group) wereadministered vehicle, 3 mg/kg of the positive control FGF21-Fc, 1.5mg/kg of scFc-GDF15, or 1.5 mg/kg of FcΔ10(−)-(G4S)4-GDF15:FcΔ10(+,K)weekly for six weeks, followed by a five-week washout. Body weight andtriglyceride levels were determined. Naive male spontaneously obesecynomolgus monkeys were prescreened for health and had a body massindex >41. Monkeys were acclimated to single housing, experimentalprocedures and handling for 6 weeks prior to treatment. Monkeys weresorted into 4 groups receiving once weekly SC injection for 6 weeks(days 0, 7, 14, 21, 28, and 35) for each group to have similar baseline.Overnight fasting blood samples were collected at pre-dose days -24, -17and -10, and on days 6, 13, 20, 27, 34, and 41 (6 days after each weeklydose) during the treatment phase. During the washout phase, bloodsamples were collected on days 48, 55, 62, 69 and 76. Body weight wasmeasured once a week and food intake was monitored daily for each monkeythroughout the study. Each monkey received unlimited feed for a limitedamount of time (1 hour) at the morning and evening feeding,approximately 8 hours apart. A 150 g apple snack, for a limited amountof time (1 hour), was provided between meals. The remaining food orapple was removed and weighed after each meal or snack to calculate foodintake.

GDF15-Fc fusion proteins reduced body weight (FIG. 1) and triglyceridelevels (FIG. 2), similar to FGF21-Fc.

Example 2: GDF15(WT)-Linker-Fc Molecule Attributes

The FcΔ10(−)-(G4S)4-GDF15 (SEQ ID NO: 39) molecule as described inExample 1 and FcΔ10(+)-(G4)-GDF15 (SEQ ID NO: 40) were analyzed forattributes that may affect its stability and manufacturability (e.g.,for commercial manufacturing). The GDF15 molecules (e.g.,FcΔ10(−)-(G4S)4-GDF15 and FcΔ10(+)-(G4)-GDF15) were determined to behighly heterogeneous (e.g., analysis of an ion exchange column fractionof FcΔ10(−)-(G4S)4-GDF15 shows the molecule is highly heterogeneous,FIG. 3), an undesirable feature for manufacturability of a molecule. Todetermine the attributes of the GDF15 molecules that result in a highlyheterogenous population, analysis of the molecules by size exclusionchromatography, SDS PAGE gel, and mass spectrometry was performed. Alack of difference in retention time by size exclusion chromatographyindicated that aggregation or gross degradation are unlikely to beresponsible for the heterogeneity. There was also a lack of differenceon an SDS PAGE gel, which indicated that disulfide mispairing or grossdegradation are also unlikely to be responsible for the heterogeneity.

MS analysis was also performed to evaluate the heterogeneity ofFcΔ10(−)-(G4S)4-GDF15 (SEQ ID NO: 39). The GDF15 molecule was purifiedusing mono S, 1 ml column and fraction number 25 (P1), fraction number28 (P2), and fraction number 31 (P3) (FIG. 3) were collected andsubmitted for MS analysis. About 50 μg of the fractions were dried down,resuspended in 25 μL of 150 mM Tris, pH 7.5/8M urea/40 mMhydroxylamine/10 mM DTT, and then incubated for 1 hour at 37° C. Thesamples were alkylated with 20 mM iodoacetamide (IAM) for 30 minutes atroom temperature in the dark. The samples were then diluted to 100 μLwith water and 2 μg of trypsin (1:25) and digested overnight at 37° C.The digests were acidified, followed by injection onto a Waters(Milford, Mass.) NanoAcquity UPLC system. Samples were first loaded ontoa 180 μm×20 mm Symmetry C18 trapping column at 15 μL/min, followed bypeptide separation on an Agilent (Santa Clara, Calif.) Zorbax 0.5 mm×250mm 300SB-C18column. Buffer A was 0.1% formic acid/water, while buffer Bwas 0.1% formic acid/ 99.9% acetonitrile. The gradient consisted ofinitial conditions at 1% B, followed by an increase to 45% B over 85minutes, to 97% B over 1 minute, isocratic at 97% B for 6 minutes, to 1%B over 3 minutes, and then isocratic at 1% B for 20 minutes. The UPLCcolumn effluent was sprayed into a Thermo Fisher Scientific (San Jose,Calif.) Orbitrap Velos Pro mass spectrometer using the standard heatedelectrospray ionization II (HESI II) ionization source. The massspectrometer method consisted of a full MS scan of m/z [300-2000] at 30Kresolution, followed by MS/MS (CID activation) of the top 10 mostabundant precursor ions. The following instrument parameters were usedfor the analysis: source voltage=3.5 kV; capillary temperature=275° C.;S-lens RF level=50%; activation time=10 msec; normalized collisionenergy=35; isolation width=2.0 Da; and threshold=1.0E4. The Xtractcomponent of the Thermo Xcalibur 2.1 software was used for deconvolutionof high-resolution MS data. Averaged data from [300-2000] weredeconvoluted using a S/N threshold of 1.2 and a resolution of 100,000 atm/z 400. Deconvoluted peptide masses (glycosylated and non-glycosylated)were displayed as monoisotopic [M+H]⁺. The various glycosylated specieswere confirmed by the stepwise loss of glycan subunits and the presenceof the unglycosylated precursor ion as the most intense fragmentfollowing CID activation.

The MS results showed that the varying degrees of deamidated species(e.g., 70% of P1, 47% of P2, and 24% of P3) and glycosylationdistribution (mostly monosaccharide and trisaccharide) on the linkercontributed to highly heterogeneous nature of the GDF15 molecule asshown in its CEX profile (FIG. 3). It was determined that the (G4S)4linker (e.g., present in FcΔ10(−)-(G4S)4-GDF15) was highly glycosylatedand phosphorylated, with varying degrees and types of glycosylationand/or phosphorylation, and the N-terminus of the active fragment ofwildtype human GDF15 was highly susceptible to deamidation andisomerization (see e.g., FIG. 4, which shows certain masses extractedfrom the full mass spec data that correspond to the unmodified,deamidated, and isomerized species of the peptide that containsasparagine at position 3. The extracted masses were m/z [590.25-590.75]from the doubly charged versions of the peptide). The asparagine atposition 3 (in reference to SEQ ID NO: 6, the amino acid sequenceencoding the active fragment of hGDF15) was highly susceptible todeamidation and isomerization and the aspartate at position 5 (inreference to SEQ ID NO: 6, the amino acid sequence encoding the activefragment of hGDF15) was highly susceptible to isomerization.

Based on these attributes, manufacturing a generally homogenouspopulation of a GDF15-Fc fusion protein having the active fragment ofwild type human GDF15 with a linker to the Fc region (e.g., forcommercial manufacturing) would be challenging.

Example 3: Activity of GDF15-Fc Fusion Proteins Without a Linker

To address the heterogeneity issues described in Example 2, new GDF15-Fcfusion proteins that 1) eliminated the linker between the GDF15 regionand the Fc region and 2) eliminated or substituted the N-terminalresidues of the active fragment of wild-type human GDF15 (e.g.,GDF15(Δ3) (SEQ ID NO: 13), where the first three amino acids of theactive fragment of wild type human GDF15 is deleted, or GDF15(N3D) (SEQID NO: 16), in which the asparagine at position 3 of the active fragmentof wild type human GDF15 is mutated to aspartate).

In addition to the charged pair mutation in the Fc region of theGDF15-Fc fusion protein and the Fc molecule for the non-covalentassociation of the Fc molecule to the Fc region of the GDF15-Fc fusionprotein to form a heterodimer, some of the new molecules were designedto also include an interchain disulfide bond in the CH3 region, or“cysteine clamp” (molecules that include “CC” in their designation) toaugment the heterodimerization of the GDF-Fc molecule with an Fcmolecule.

Four new GDF15-Fc fusion proteins in which 1) the linker between theGDF15 region and the Fc region was deleted and 2) the N-terminalresidues of GDF15 were eliminated or substituted were generated. In twoof the four molecules, an interchain disulfide bond was also introducedinto the CH3 domain of the Fc region of the GDF15-Fc fusion protein (aswell as its corresponding Fc molecule for heterodimerization). Thepotency and pharmacokinetic (PK) properties of these molecules(FcΔ10(−)-GDF15(Δ3) (SEQ ID NO: 41); FcΔ10(−)-GDF15(N3D) (SEQ ID NO:42); FcΔ10(−,CC)-GDF15(Δ3) (SEQ ID NO: 43); FcΔ10(−,CC)-GDF15(N3D) (SEQID NO: 44)) were compared to the earlier generationFcΔ10(−)-(G4S)4-GDF15 (SEQ ID NO: 39), in mice.

To determine the potency of the molecules, food intake was determined.Seven to eight-week old single-housed male ob/ob mice were sorted intodifferent treatment groups with each group having comparablepretreatment body weight and food intake levels. Animals were treatedwith 0.32 ug/kg, 1.6 ug/kg, 8 ug/kg, 40 ug/kg, 0.2 mg/kg, 1 mg/kg, or 5mg/kg of a GDF15-Fc fusion protein (a dimer ofFcΔ10(−)-GDF15(Δ3):FcΔ10(+,K) (SEQ ID NOs: 41 and 32);FcΔ10(−)-GDF15(N3D):FcΔ10(+,K) (SEQ ID NOs: 42 and 32); orFcΔ10(−,CC)-GDF15(Δ3):FcΔ10(+,K,CC) (SEQ ID NOs: 39 and 32)) throughsubcutaneous injection, and overnight food intake was measured. Datapresented is an average of 2-4 independent studies (FIG. 5). The fournew molecules, FcΔ10(−)-GDF15(Δ3) (SEQ ID NO: 41); FcΔ10(−)-GDF15(N3D)(SEQ ID NO: 42); FcΔ10(−,CC)-GDF15(Δ3) (SEQ ID NO: 43); andFcΔ10(−,CC)-GDF15(N3D) (SEQ ID NO: 44), had comparable potency as theearlier generation GDF15-Fc fusion protein, FcΔ10(−)-(G4S)4-GDF15 (SEQID NO: 39).

To determine the pharmacokinetics of the molecules, 18-wk old malediet-induced obese C57B1/6 mice were dosed with 1 mg/kg proteinsubcutaneously, and serial sampling was performed at 1, 4, 8, 24, 72,168, 240, and 336 hr post-dose. The four new molecules,FcΔ10(−)-GDF15(Δ3) (SEQ ID NO: 41); FcΔ10(−)-GDF15(N3D) (SEQ ID NO: 42);FcΔ10(−,CC)-GDF15(Δ3) (SEQ ID NO: 43); and FcΔ10(−,CC)-GDF15(N3D) (SEQID NO: 44), had comparable pharmacokinetic properties as the earliergeneration GDF15-Fc fusion protein, FcΔ10(−)-(G45)4-GDF15 (SEQ ID NO:39) (FIG. 6).

Example 4: Further Engineering of GDF15-Fc Fusion Proteins Without aLinker

As the newly designed molecules with improved manufacturability andstability attributes had similar potency and PK properties as theearlier generation molecule, the molecules were further engineered toreduce possible heterogeneity and reduce Fc effector function andincrease potency.

To further reduce heterogeneity of the GDF15 region, instead ofsubstituting the asparagine at position 3 with aspartate, the asparaginewas substituted with glutamine. In addition, the molecules wereengineered to have two changes introduced in the N-terminus of GDF15,e.g., GDF15(Δ3/D5E) (SEQ ID NO: 17), GDF15(N3Q/D5E) (SEQ ID NO: 18) toeliminate the high rate of deamidation and isomerization of the nativeGDF15 protein. To reduce Fc effector function by and improve potency,the molecules were also engineered to have the hinge region of the Fcregion deleted further by having an additional six amino acids deletedfrom the Fc hinge region (e.g., FcΔ16 instead of FcΔ10) to decreasebinding to FcγR. The same engineering of the hinge region was performedfor the corresponding Fc molecules to which the GDF15-Fc fusion proteinsheterodimerize with.

The activity of the further engineered GDF15-Fc fusion proteins,FcΔ16(−,CC)-GDF15(Δ3/D5E) (SEQ ID NO: 45), FcΔ16(−,CC)-GDF15(N3Q/D5E)(SEQ ID NO: 46), and FcΔ16(−)-GDF15(N3Q/D5E) (SEQ ID NO: 47), weretested in cynomologus monkeys. Naive male spontaneously obese cynomolgusmonkeys were acclimated/trained to procedural manipulations (e.g., bloodcollection, subcutaneous injection, body weight measurement, feedingschedule) for 10 weeks prior to treatment initiation. Eighty (80)monkeys were sorted into 8 treatment groups of n=10 monkeys each basedon data collected during acclimation/training phase (blood chemistriesand body weight). Each treatment group was administered vehicle, 3 mg/kgof the positive control FGF21-Fc, 0.5 mg/kg of FcΔ16(−,CC)-GDF15(Δ3/D5E)(along with its heterodimerization partner, FcΔ16(+,K,CC) (SEQ ID NO:35)), 3.0 mg/kg of FcΔ16(−,CC)-GDF15(Δ3/D5E) (along with itsheterodimerization partner, FcΔ16(+,K,CC) (SEQ ID NO: 35)), 0.5 mg/kg ofFcΔ16(−,CC)-GDF15(N3Q/D5E) (along with its heterodimerization partner,FcΔ16(+,K,CC)), 3.0 mg/kg of FcΔ16(−,CC)-GDF15(N3Q/D5E) (along with itsheterodimerization partner, FcΔ16(+,K,CC)), 0.5 mg/kg ofFcΔ16(−)-GDF15(N3Q/D5E) (along with its heterodimerization partner,FcΔ16(+,K) (SEQ ID NO: 36)), or 3.0 mg/kg of FcΔ16(−)-GDF15(N3Q/D5E)(along with its heterodimerization partner, FcΔ16(+,K) (SEQ ID NO: 36)).Subcutaneous injections of each were given once a week for 4 weeksduring the treatment phase followed by a 4-week washout phase; bloodcollection and body weight monitoring occurred weekly and food intakeoccurred daily during treatment and washout phases. The graph representsn=5-6/group and data are represented as group means±SEM. Statisticalanalysis was performed by ANCOVA and statistical significance is denotedas *p<0.05, **p<0.01 and ***p<0.001 versus vehicle. Monkeys with rapiddrug clearance were suspect of anti-drug antibodies (ADAs) and wereexcluded from analysis.

Unexpectedly, the newly engineered GDF15-Fc fusion proteins lost almostall potency (FIG. 7). None of the newly engineered GDF15-Fc fusionproteins reduced body weight to a similar degree as to FGF21-Fc, incontrast to the previously generated GDF15-Fc fusion proteins (seeExample 1, FIG. 1)

Example 5: Restoration of GDF15-Fc Fusion Protein Activity inCynomologus Monkeys

The GDF15-Fc fusion proteins in Example 4 as compared to the GDF15-Fcfusion protein in Example 1 had the following differences as shown inTable 7:

TABLE 7 Differences between GDF15-Fc Fusion Proteins in Examples 1 and 4GDF15-Fc Molecules in GDF15-Fc Molecules in Example 1: Example 5:Efficacious in Not Efficacious in Cynomologus Monkeys CynomologusMonkeys Δ10 in Fc region Δ16 in Fc region No cysteine clamp Cysteineclamp Has linker No linker Wild type GDF15 Two mutations in N-terminusof GDF15

To restore potency, different aspects of the molecules that wereefficacious in the monkeys were re-introduced into new GDF15-Fc fusionproteins. The cysteine clamp (CH3 interchain disulfide bond) waseliminated and a linker reintroduced for FcΔ16(−)-(G4Q)4-GDF15(N3Q) (SEQID NO: 49); FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) (SEQ ID NO: 50) andFcΔ16(−)-G4S-GDF15(N3Q/D5E) (SEQ ID NO: 54). However, the linker used inthis Example cannot be glycosylated (e.g., G4Q) or was shorter (G4Sinstead of (G4S)4), to reduce glycosylation. Also, forFcΔ16(−)-(G4Q)4-GDF15(N3Q), the mutation at position 5 was eliminated.Lastly, for the new molecule FcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E)(SEQ ID NO: 57), the smaller deletion of the hinge region of the Fcregion was reintroduced, however with L234A/L235A mutations in the Fcregion, which should eliminate FcγR binding.

These new molecules were compared to FcΔ10(−)-(G4S)4-GDF15, which wasshown to be efficacious in cynomologus monkeys in Example 1. Naive malespontaneously obese cynomolgus monkeys were acclimated/trained toprocedural manipulations (e.g., blood collection, subcutaneousinjection, body weight measurement, feeding schedule) for 2 weeks priorto treatment initiation. Forty-two (42) monkeys were sorted into 6treatment groups of n=7 monkeys each based on data collected duringacclimation/training phase (blood chemistries and body weight). Eachtreatment group was administered vehicle, 1.5 mg/kg ofFcΔ10(−)-(G4S)4-GDF15 (along with its heterodimerization partner,FcΔ10(+,K)), 1.5 mg/kg of FcΔ16(−)-(G4Q)4-GDF15(N3Q) (along with itsheterodimerization partner, FcΔ16(+,K)), 1.5 mg/kg ofFcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) (along with its heterodimerizationpartner, FcΔ16(+,K)), 1.5 mg/kg of FcΔ16(−)-G4S-GDF15(N3Q/D5E) (alongwith its heterodimerization partner, FcΔ16(+,K)), or 1.5 mg/kg ofFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) (along with itsheterodimerization partner, FcΔ10(+,K,L234A/L235A) (SEQ ID NO: 37).Subcutaneous injections were given once a week for 2 weeks during thetreatment phase; blood collection and body weight monitoring occurredweekly and food intake was monitored daily during the treatment phase.The graph represents n=7/group and data is represented as groupmeans±SEM. Statistical analysis was performed by ANCOVA and statisticalsignificance is denoted as *p<0.05, **p<0.01 and ***p<0.001 versusvehicle. The new molecules restored potency (FIG. 8).

Based on these results, the N3Q mutation was determined to not impactthe GDF15 activity in the monkeys, and that the double mutation in GDF15(N3Q/D5E) also did not impact GDF15 activity in the monkeys. The16-amino acid Fc hinge deletion (Δ16) was also shown to have a similareffect as the 10-amino acid Fc hinge deletion (Δ10) in the monkeys.Lastly, the linker was shown to be a critical component for activity inthe monkeys. Though whether the linker is a G4S or G4Q does not affectactivity, the length of the linker is important for activity. The longerlinkers (e.g., (G4S)4 and (G4Q)4 in FIG. 8) are more potent as comparedto a shorter linker (e.g., G4S).

Example 6: Food Intake Assay in ob/ob Mice forFcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) andFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E)

A food intake assay was used to evaluate efficacy of two differentGDF15-Fc fusion proteins. Seven to eight weeks-old single-housed maleob/ob mice were sorted into different treatment groups (n=5 per group)with each group having comparable pretreatment body weight and foodintake levels. Animals were treated with 0.32 ug/kg, 1.6 ug/kg, 8 ug/kg,40 ug/kg, 0.2 mg/kg, 1 mg/kg, or 5 mg/kg of the heterodimerFcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E):FcΔ16(+,K) orFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E):FcΔ10(+,K,L234A/L235A)through subcutaneous injection, and overnight food intake was measured.The results of a representative experiment for each GDF15-Fc fusionprotein is shown in a dose response curve forFcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) (FIG. 9) andFcΔ10(−,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) (FIG. 10). The results showboth GDF15-Fc fusion proteins reduce food intake in acute ob/ob mice.The ED50 in this assay is shown in Table 8.

TABLE 8 ED50 in Food Intake Assay ED50 (mg/kg) Molecule n = 3-5FcΔ16(−)-(G4Q)4-GDF15(N3Q/D5E) 7.4 ± 4.2 FcΔ10(-,L234A/L235A)-(G4Q)4-GDF15(N3Q/D5E) 5.8 ± 0.7

While the present invention has been described in terms of variousembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed. In addition, the section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

All references cited in this application are expressly incorporated byreference herein for any purpose.

What is claimed is:
 1. A fusion protein comprising a GDF15 region joinedto an Fc region via a linker, wherein the GDF15 region comprises theamino acid sequence of SEQ ID NO: 6, except for having mutations at theasparagine residue at position 3 of SEQ ID NO: 6 and the aspartateresidue at position 5 of SEQ ID NO: 6, wherein the aspartate at position5 of SEQ ID NO: 6 is substituted with glutamate, and wherein the linkeris a (G4Q)n linker, wherein n is greater than
 2. 2. The fusion proteinof claim 1, wherein n is 3 or
 4. 3. The fusion protein of claim 2,wherein n is
 4. 4. The fusion protein of claim 1, wherein the asparagineat position 3 is substituted with glutamine.
 5. The fusion protein ofclaim 1, wherein the GDF15 region comprises the amino acid sequence ofSEQ ID NO:
 18. 6. The fusion protein of claim 5, wherein the Fc regioncomprises a charged pair mutation.
 7. The fusion protein of claim 5,wherein the Fc region comprises a truncated hinge region.
 8. The fusionprotein of claim 1, wherein the Fc region comprises the amino acidsequence of SEQ ID NO:
 30. 9. A fusion protein comprising the amino acidsequence of SEQ ID NO:
 50. 10. A dimer comprising a fusion proteincomprising the amino acid sequence of SEQ ID NO: 50 and a proteincomprising the amino acid sequence of SEQ ID NO:
 36. 11. A tetramercomprising the dimer of claim 10.